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EXPERIMENTS IN THE ARTIFICIAL PROPAGATION 
OF FRESH-WATER MUSSELS ^ ^ ^ > 



From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXVIII, 1908 
Proceedings of the Fourth International Fishery Congress : Washington, 1908 




WASniNGTON :::::: GOVERNMENT PRINTING OFFICE 




Pass O LI Olt^_ 
Book ' L ^ 



EXPERIMENTS IN THE ARTIFICIAL PROPAGATION 
OF FRESH-WATER MUSSELS ^ ^ ^ ^ 



From BULLETIN OF THE BUREAU OF FISHERIES, \'olmne XXVIII, 1908 



Proceedings of ihe Fourth International Fishery Congress 



Washington , i<;io8 




I'cc-vx 



WASHINGTON :::::: GOVERNIMEXT PRINTING OFFICE 






BUREAU OF FISHERIES, DOCUMENT NO. 671 
Issued April. 1910 



EXPERIMENTS IN THE ARTIFICIAL PROPAGATION 

OF FRESH-WATER MUSSELS 

,* 

By George Lefevre and W. C. Curtis 

Professors of Zoology, Uyiiversity of Missouri 
J- 

Paper presented before the Fourth International Fishery Congress 
held at Washington, U. S. A., September 22 to 26, 1908 



CONTENTS. 

Page. 

Object and scope of the undertaking 617 

Some facts in the reproduction and Ufe history of mussels 618 

Breeding seasons ■_ 620 

The larva, or glochidium 62 1 

Implantation of the glochidia 622 

Experimental infections 622 

Method 622 

Behavior of the fishes — Their susceptibility and resistance 623 

Duration of the parasitic period 624 

Post-larval growth of mussels 625 

Infection of large numbers of fish 626 

616 



EXPERIMENTS IN THE ARTIFICIAL PROPAGATION 
OF FRESH -WATER MUSSELS. 



By GEORGE LEFEVRE and \V. C. CURTIS, 
Professors of Zoology, University of Missouri. 



OBJECT AND SCOPE OF THE UNDERTAKING. 

The threatened extinction in the Mississippi River and its more important 
tributaries of those species of mussels whose shells have been taken in enormous 
numbers during the past fifteen years for the manufacture of pearl buttons has led 
the United States Bureau of Fisheries to undertake an extensive investigation 
to determine the possibility of artificial propagation of these mollusks, and to 
devise such means as may be practicable for restocking depleted waters which 
present favorable conditions. The general direction of the investigation has 
been placed in the hands of the writers, who for the past three years have 
devoted to the work as much time as their professional duties would allow. 
In certain phases of the work, however, many others have collaborated. 

It was recognized at the outset that if the investigation was to have real 
practical value it would have to be wide in scope and extend over a period of 
at least several years. At that time little was known with accuracy of the 
breeding habits and seasons of the commercial species, of the biological and 
physical conditions under which they live, of their distribution, and of many 
other essential matters, while it yet remained to be discovered whether artificial 
propagation could be successfully accomplished. At the verj' inception of the 
work, therefore, a comprehensive plan was outlined which was designed to 
include every subject that might bear even remotely upon the central problem, 
and, although many parts of this programme have not yet been touched, much 
progress has been made in some of the more important lines. 

The plan of work contemplated, besides a thorough investigation of artificial 
propagation, a detailed study of the life history and ecology of the Unionidae, 
emphasizing especially the geographical distribution of the group throughout the 
Mississippi Valley, the breeding habits and seasons, the physical conditions of 
the waters in which the different species tlirive, food, enemies, and diseases, 

617 



6l8 BULLETIN OF THE BUREAU OF FISHERIES. 

rate of growth, post-embryonic development, and the mutual behavior of 
glochidia, or larvse, and fishes as parasites and hosts. 

The results that have already been obtained, although far from being 
complete, will serve as a basis for future investigations, while the lines of attack 
in the main problems have been definitely indicated. We have proceeded far 
enough to make it clear that the ultimate-end of the investigation is assured, 
and with proper facilities for the infection and care of large numbers of fish 
the final success of the work is no longer in doubt. The essential facts in the 
life history of the Unionidse are known ; the general conditions of infection have 
been determined; the entire feasibility of artificially infecting large numbers of 
fish has been established; while the requisite conditions for placing artificial 
propagation on a practical basis are clearly understood. Furthermore, during 
the summers of 1907 and 1908 several parties, working under the direction of 
the Bureau of Fisheries, have made extensive observations on a number of 
different rivers in the upper Mississippi Valley, with a view to determining the 
distribution of all of the species of mussels occurring in these waters. The 
rivers that have been explored in this manner are the upper Mississippi, Illinois, 
Iowa, Minnesota, Wabash, Ohio, and Tippecanoe, as well as several smaller 
streams in Indiana. When the results obtained by these parties have been 
brought together, we should be in possession of detailed information regarding 
the distribution and relative abundance of at least all of the important com- 
mercial species throughout this region. In addition, the breeding seasons of 
almost all of the important commercial species have been determined, and a 
large mass of miscellaneous data relating to a great variety of subjects has 
been accumulated. 

Some of the more important results that have thus far been obtained in 
the investigations are briefly presented here. 

SOME FACTS IN THE REPRODUCTION AND LIFE HISTORY OF MUSSELS. 

As is well known, the Unionidse carry their young in the gills, which act 
as a marsupium or brood chamber until the completion of the embryonic 
development. The eggs are passed from the ovaries into the suprabranchial 
chambers, where they are fertilized by spermatozoa brought in by the inhalent 
current of water. In many species the eggs are bound together in masses by a 
gelatinous or glutinous matrix which is secreted by the gills and which may be 
very tenacious. This, however, in most cases gradually dissolves away as 
development proceeds, until at the close of the embryonic period the larvse or 
glochidia are no longer agglutinated. These masses assume in different species 
many forms of flat plates or cylindrical cords which fill the water tubes of 
the gills. The latter may be greatly distended or modified in various ways 
to receive the egg-masses. In some species, on the other hand, the condition 



PROPAGATION OF FRESH-WATER MUSSELS. 619 

just described is absent and the eggs are either not agglutinated at all or are 
only loosely held together in a slightly viscid substance. 

The extent to which the gills are modified as a marsupium varies greatly in 
different genera. In many, as in Anodonta, Pleurobema, Strophitus, Symphynota, 
and Unio, the entire outer gill of each side is thus utilized and when filled with 
embr}^os or glochidia, presents a much swollen, padHke appearance. In others, 
for example, Lampsilis, Obovaria, Plagiola, only the posterior portion, varying 
from one-third to two-thirds of the entire outer gill, is used as a receptacle for 
the eggs, a sharp demarcation existing between the marsupial region and the 
anterior respiratory part. The former is highly modified in structure and may 
be readily recognized, even when empty. In Obliquaria reflexa a var^'ing 
number of enlarged and distended water tubes contain the dense, glutinous 
cords in which the embryos are embedded. Finally, there are at least two 
genera, namely, Ouadrula and Tritigonia, in which all four gills are utilized as 
brood chambers. This condition would seem to be the most primitive one, as 
the gills show but little structural modification and when empty are indistin- 
guishable from those of the male mussel. 

In all of the species of the genus Lampsilis which we have examined the 
posterior margin of the valves in the female is less pointed than in the male, and 
by reason of this difference in the shape of the shells the sexes may be readily 
distinguished externally. In most of the other genera that have come under 
our observation, however, the shells of the two sexes are identical, and an inter- 
nal examination is necessary in order to determine the sex of an individual. 

The entire embryonic development takes place within the marsupium, and 
at the end of this period the glochidium, which has become liberated from the 
egg membrane within the gill, is fully formed. In those species which are 
known as "summer breeders," the glochidia are discharged at once, while in 
many others, the so-called "winter breeders," they are carried in the gills for 
several months before being set free in the water. 

The glochidia, as has long been known, do not complete their development 
unless they become attached to fishes, and the metamorphosis occurs while 
they are living parasitically in a cyst formed by the epidermis of the host. 
After discharge from the marsupium, the glochidia under natural conditions 
lie quiescent on the bottom, their valves gaping widely apart, and unless chance 
brings them in contact with a fish they will die. Once attached to the gills or 
external parts of a fish, however, the rest of the development, if conditions are 
favorable, is assured. 

This unique peculiarity in the life history of fresh-water mussels has been 
taken advantage of in artificial propagation, as it has been found that a single 
fish, even though small, may successfully carry to the close of metamorphosis 
several hundred glochidia. One fish may, therefore, be made to do the work 



620 BULLETIN OF THE BUREAU OF FISHERIES. 

of a large number in a state of nature, for under ordinary conditions it is 
extremely improbable that more than a very 'few glochidia would succeed in 
attaching themselves to a single fish. 

After a parasitic life of variable length, during which the metamorphosis 
is completed, the cyst breaks down, and the young mussel, thus liberated, falls 
to the bottom and leads thereafter an independent life. 

BREEDING SEASONS. i 

We have confirmed the conclusion, first reached by Sterki (Nautilus, vol. 
IX, 1895), that the Unionidfe, with respect to their breeding seasons, fall into 
two classes, the so-called "summer breeders" and "winter breeders." The 
latter designation, however, is not strictly appropriate, for in the species which 
belong to this group the eggs are fertilized during the latter part of the summer, 
usually in August, and the glochidia, which are carried in the brood chambers 
over the winter, are not discharged until the following spring and summer. In 
fact, in some cases the close of one breeding period may overlap on the begin- 
ning of the next, as one may still find in late July some females gravid with 
glochidia, while in others the eggs are passing into the gills for the next season. 
In the case of the summer breeders the eggs are fertilized during the late spring 
and summer, and the glochidia are in all probability discharged before the end 
of August, or certainly not later than the middle of September. In the species 
of this class of which we have the completest records, ovulation begins in May 
and early June and may last well into August, but after the end of August 
gravid females have not been found. 

In view of the above facts, it would seem in better accord with actual 
conditions to separate the species with respect to the length of time that the 
glochidia remain in the gills, designating them as those which have a "short 
period" of gravidity and those having a "long period," rather than to dis- 
tinguish them as "summer breeders" and "winter breeders," respectively, for 
in the case of the latter neither ovulation nor discharge of the glochidia takes 
place in winter. The breeding season is undoubtedly a generic character, for, 
so far as our observations go, all species of a given genus belong to the same 
class. 

The following list shows the distribution, with respect to the period of 
gravidity, of the genera we have had under observation: 

Long period: vShort period: 

Alasmidonta. Obliquaria. 

Anodonta. Pleurobema. 

Lampsilis. Quadrula. 

Obovaria. Tritigonia. 

Plagiola. Unio. 
Strophitus. 
Symphynota. 



PROPAGATION OF FRESH-WATER MUSSELS. 621 

As there are important commercial species in each class, the futility of any 
law which would prohibit the taking of mussels during their breeding season 
becomes at once apparent, for there is no month in the year when some species 
are not carrying either embryos or glochidia. 

THE LARVA, OR GLOCHIDIUM. 

Two well-marked types of glochidia occur in the Unionidae, one provided 
with stout hooks on the ventral margin of the valves and the other entirely 
hookless. The former are characteristically parasitic on the fins and other 
external parts of the fish, the latter on the gills. We have given special atten- 
tion to a study of the physiological peculiarities correlated with these two types 
of structure, including their differences in behavior in attachment. 

The occurrence of the two types of glochidia in the genera which we have 
examined is shown below. 

Hookless glochidia: Hooked glochidia: 

Lampsilis (except L. Isvissimus). Anodonta. 

Obliquaria. Lampsilis Isevissimus. 

Obovaria. Strophitus. 

Plagiola. Symphynota. 

Pleurobema. 
Quadrula. 
Tritigonia. 
Unio. 

It is thus seen that the hookless type is by far the commoner one, and 
furthermore, practically all of the commercial species of mussels belong to 
the genera included in this class. 

The type of glochidium is constant for the genus, so far as our observations 
have gone, except in the genus Lampsilis, one species of which, L. leevissimus, 
produces glochidia having hooks, while all of the remaining species that we 
have examined have the hookless forms. The glochidia of L. IcBvissimus , more- 
over, are of quite a different form from those of any other species we have 
encountered, being shaped like an axe head when seen in side view. 

The hookless glochidia are usually much smaller than those having hooks, 
and also differ characteristically from the latter in shape. 

We have repeatedly determined the fact that the hooked glochidia form 
permanent attachment as a rule only to external parts of the fish, as the margins 
of the fins, operculum, anus, and mouth, the barbules, or even to scales and 
along the fin rays occasionally. Wherever a free border is presented that may 
be grasped between the valves of the glochidium, attachment is possible, the 
hooks holding securely as soon as the valves have closed. Hooked glochidia, 
furthermore, are not infrequently found on the gill arches and rakers, as these 
places furnish the requisite conditions for a firm grasp. Glochidia of this type, 



622 BULLETIN OF THE BUREAU OF FISHERIES. 

however, do not as a rule acquire a permanent lodgment on the delicate gill 
filaments, as their hooks usually tear througfi these when grasping. Their 
greater size would also seem to render it difficult for the hooked glochidia to 
become properly embedded under the epidermis of the slender filament. 

Hookless glochidia, on the other hand, are only rarely found on the external 
parts of the fish, and although they become attached readily to such places, in 
the absence of hooks their grasp is insecure, and the usual consequence is that 
they are all soon brushed off". Being hookless and quite small, they are admi- 
rably adapted to lodgment on the gill filaments, among which they may become 
entangled in great numbers. 

Interesting differences of a physiological nature also are correlated with 
the two structural types, apparently in adaptation to their different parasitic 
habits. 

IMPLANTATION OF THE GLOCHIDIA. 

Contact with any part of the fish affords the stimulus which causes the 
adductor muscle of the glochidium to contract and close the valves. This stim- 
ulus is purely a mechanical one, as the same result may be produced by touch- 
ing with any object. In this way they may be caused to snap shut on a bristle 
or hair, or on the edge of a piece of paper. Once attached to the fish, how- 
ever, the glochidium exerts a stimulus upon the epidermal cells of the host 
which causes them to undergo a rapid proliferation. The stimulus in this case 
is undoubtedly a chemical one, and its immediate effect is to throw the cells 
of the epidermis into active mitotic division. The epidermis now grows rapidly 
over the glochidium, which may become completely embedded in three hours 
after attachment in the case of the gill parasites. The proliferation of the epi- 
dermis of the fins occurs more slowly and in this situation twelve to twenty- 
four hours are usually required before the glochidia are covered. 

After the process of implantation is completed, the glochidia are entirely 
inclosed within the cellular cyst, where they are nourished by the host until 
liberated by the rupture of the cyst at the end of the parasitic period. It is 
during this stage of the development that the metamorphosis occurs and the 
structure of the glochidium is replaced by that of the mussel. 

EXPERIMENTAL INFECTIONS. 
METHOD. 

A very large number of experimental infections have been made, and the 
conditions determining infection have been investigated as far as the material 
at our disposal has allowed. This study has included not only the behavior of 
both types of glochidia, their manner and place of attachment, but the reac- 
tions of the fish as well. 



PROPAGATION OF FRESH-WATER MUSSELS. 623 

Twenty different species of mussels, yielding both hooked and hookless 
glochidia, have been used for the infections. The majority of these have been 
species showing the long period of gravidity, although several of the summer 
breeding species have also been employed. Eighteen species of fishes have 
been used, and their reactions have been carefully obser\-ed, but, as all are 
not equally favorable for receiving and carrj^ing the parasites, the greater num- 
ber of infections have been made upon five or six species. 

When only a few fishes are to be used at a time, the method which has been 
employed is quite simple. The brood chambers are excised from the gravid 
mussels, split open lengthwise, and the ripe glochidia washed and scraped out 
into dishes of water. The fishes are then placed together with the glochidia in 
small aquaria, and, if the infection is to be made with gill parasites, the water 
is agitated by mechanical means, in order to keep the glochidia suspended and 
thus insure their being taken into the mouth of the fish. When using hooked 
glochidia for external attachment, however, it is better not to stir them up, as 
the fishes usually settle down to the bottom of the vessel and, by rubbing their 
fins among the glochidia, quickly become infected to any desired degree. 

When making gill infections, the fishes must be examined at inter^^als of 
one or two minutes for the purpose of observing the rapidity with which the 
glochidia are attaching, and when the degree is reached which experience has 
shown to be the optimum for the species of fish in question, the exposure is at 
once discontinued. Usually the gill infections take place very quickly, and 
in four or five minutes the fishes will have received as many glochidia as they 
can safely carry, although of course the duration of exposure depends upon 
the abundance of glochidia in the water. Other things being equal, the fewer 
glochidia present the more prolonged the exposure must be. Experience has 
shown that the best results are obtained when the glochidia are used in ven,' 
"dilute mixtures," for it is difficult to avoid overinfection, if they are at all 
concentrated. Some species of fishes, however, are less susceptible than others, 
and a longer exposure is necessary to insure the attachment of an adequate 
number of glochidia. In certain cases we have had to allow the fishes to 
remain with the glochidia for from two to three hours, or even longer, in order 
to obtain a satisfactory degree of infection. 

In nearly all of our experiments we have used young fishes, from three to 
six inches in length, as for many reasons they are more convenient than larger 
ones. 

BEHAVIOR OF FISHES — THEIR SUSCEPTIBILITY AND RESISTANCE. 

The fishes when first placed in contact with glochidia give evidence of great 
irritation, and as soon as the gills begin to be infected they exhibit violent and 
rapid breathing movements, apparently in the attempt to expel the parasites. 



624 BULLETIN OF THE BUREAU OF FISHERIES. 

After a time, however, they become quiet, and when removed from the presence 
of the glochidia behave normally, except that the more rapid respiratory move- 
ments may continue even for days afterwards. 

The fishes we have used in our experiments have shown marked differences 
in their ability to retain the glochidia both on the fins and on the gills, more 
especially on the latter. They vary even more strikingly with respect to their 
capacity for withstanding the injurious effects of gill infection, and further- 
more some fishes are undoubtedly much more susceptible than others, as the 
glochidia become attached, especially to the gills, more readily. 

Of the fishes which we have had under observation, the following species 
have proved to be the most satisfactory: rock bass, large and small mouthed 
black bass, green sunfish, and red-spotted sunfish. All of these species are very 
susceptible and readily infected with gill parasites, large numbers of which may 
be carried without apparent injmy to the fish. A single individual of any of 
the above-mentioned fishes, not more than 3 or 4 inches in length, will success- 
fully carry at least 1,000 glochidia on its gills to the close of the parasitic period, 
a number that would speedily kill many other species. 

Yellow perch, blue-gill sunfish, and crappie, although quite susceptible, are 
far less resistent to the injury and quickly succumb to overinfection. Young 
bullheads are apparently very difficult to infect, and we have not succeeded 
in causing glochidia to become attached to either their gills or fins in appre- 
ciable numbers. 

German carp are easily infected on external parts with hooked glochidia, 
but thus far all efforts to infect this fish with gill parasites have been unsuccessful. 
The glochidia will attach themselves to the gill filaments, but for some unaccount- 
able reason implantation is imperfect and they soon drop off. Apparently the 
epidermis of this fish does not react to the stimulus in such a way as to lead to 
the formation of a permanent cyst. 

In still other cases it has been found that failure is due to the fact 
that, even after complete implantation on either the gills or fins, the walls of 
the cyst slough away in a day or two and liberate the glochidia. Whether this 
is caused by unfavorable physiological conditions in the fish or in the glochidia 
we are unable to say, but there are certain facts that would seem to indicate 
that such abortive implantations are the result of a physiological immaturity 
of the glochidia, which therefore fail to call forth the necessary response on the 
part of the fish's tissues. 

DURATION OF THE PARASITIC PERIOD. 

The duration of the parasitic period is very variable. With respect to 
the glochidia of those species of mussels which remain gravid over winter the 
duration depends upon the time of year when the infection is made. Fishes 



PROPAGATION OF FRESH-WATER MUSSELS. 625 

infected in the late fall and winter will carry the glochidia for from two and a 
half to nearly four months, while in spring, when the same kind of glochidia 
are used, the metamorphosis is completed and the young clams liberated in a 
little less than a month. This great diiference in the length of time that the 
glochidia remain on the fish can not be due primarily to temperature, although 
this may have some influence, for we have observed the same differences in the 
laboratory, where the temperature of the water remains practically constant 
throughout the winter and spring. As the glochidia of the mussels which have 
the long period of gravidity are not normally discharged until spring and summer, 
it is quite possible that the fall and winter glochidia are physiologically immature, 
although capable of causing the formation of the cyst on the fish, and require 
for this reason a much longer time for the post-embryonic development. Struc- 
tiu-ally, however, they seem to be identical with those taken from the marsupium 
in the spring. 

In the case of the spring infections the young clams all leave the fish 
at about the same time, and within a very few days after the first are Uberated 
the last to go have come off. This is not true, however, of the fall and winter 
infections, for in these cases the setting free of the young mussels may be 
extended over a period of three or four weeks, or possibly more. 

We have no observations on the duration of the parasitic period in the case 
of the summer-breeding mussels. Although infections have been success- 
fully made with several of these species and kept under observ-ation for ten or 
twelve days, we have not yet had an opportunity of following the metamorphosis 
through to the end. The period is, however, undoubtedly short, probably not 
more than four weeks. Harms (Zoologischer Anzeiger, bd. xxxii, 1908) has 
recently recorded a parasitic period for two European species of Uiiio, a genus 
belonging to the group of summer breeders, of twenty-six to twenty-eight days. 

POST-LARVAL GROWTH OF MUSSELS. 

No data have been obtained on the rate of growth of fresh-water mussels 
beyond a short period after the metamorphosis, as we have not succeeded in 
keeping the young mussels alive in the laboratory for more than four weeks after 
leaving the fish. The amount of growth during that time was only a fraction 
of a millimeter. We have made, however, extensive plantings of young mus- 
sels which have been allowed to drop off the fishes in ponds and streams where 
the chances of reclaiming them in the future are very good, and it is hoped that 
by another year or two some data on the rate of gro^vth will be available. Care- 
fully measured and weighed young mussels of different sizes have also been placed 
in wire cages and set in ponds and rivers, and it is possible that additional data 
will be obtained from these experiments. 



626 BULLETIN OF THE BUREAU OF FISHERIES. 

INFECTION OF LARGE NUMBERS OF FISH. 

In order to test the feasibility of infecting large numbers of fish, which is 
a prerequisite of artificial propagation, an extensive experiment, in which 
over 25,000 fish were employed, was made at the substation of the Bureau of 
Fisheries at La Crosse, Wis., in November, 1907. Seven species of fishes and 
glochidia from four species of Lampsilis were used in the experiment. The 
fishes, with the exception of the German carp and the bullheads, were success- 
fully infected and retained in the tanks of the station for over two months, when 
they were liberated in the west channel of the Mississippi River at that place. 
The experiment completely demonstrated the possibility of making infections 
successfully on a large scale, and of retaining the fish until near the close of 
the parasitic period. 

In November, 1908, a similar experiment was carried out at the station 
of the Bureau at Manchester, Iowa, where about 12,000 large-mouthed black 
bass and crappies were infected with the glochidia of several species of Lampsilis. 
The fish were kept at the station for one month and then placed in the Maquoketa 
River between two dams. As the species of mussels used in the experiment in 
all probability do not occur in that stream, the results of the work may be 
determined if we succeed in reclaiming any of them at a future time. 

During the fall and winter of 1908-9 infected fishes were put into ponds in 
the neighborhood of Columbia, Mo., where we are absolutely certain that the 
species of mussels used do not occur, and where there is every reason for the 
expectation of subsequent reclamation. The latter experiments should yield 
valuable data on the rate of growth. 



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